Integrins are heterodimeric transmembrane glycoproteins which, inter alia, act as cell receptors for various entities, herein termed collectively “integrin ligands,” including, for example, surface molecules of other cells and extracellular matrix (ECM) proteins. Both soluble and immobilized integrin ligands are known to be ordinarily bound by integrins. Integrins are found on most types of cells. Ligand binding by integrins may result in or occur in association with a series of additional cellular events involving one or more cellular functions. These cellular events and functions, some of which are discussed below for illustrative purposes, are termed “integrin-mediated.” For a general review of integrins, see, Guidebook to the Extracellular Matrix and Adhesion Proteins (Kreis, et al, Eds.), 1993, and Pigot, et al., The Adhesion Molecule Facts Book, Academic Press, 1993.
One such integrin-mediated cellular function is signaling. For instance, certain integrins are known to transfer information from the inside to the outside of the cell (inside-out signaling) or from the outside to the inside of the cell (outside-in signaling), although other types of signaling may also occur, as may combinations thereof. An example involving inside-out signaling is the process whereby an integrin acquires or expresses affinity for ligands in response to intracellular events (integrin upregulation). Binding of integrin ligands to certain integrins (e.g., in the case of integrin-mediated cell adhesion) may initiate signal transduction events, in a manner similar to that described for other cell surface receptors. Signals thus elicited are termed outside-in signals and are involved in the regulation of various cell responses, which may include gene expression, cell differentiation, and cell proliferation.
Signaling may result in the clustering of cellular molecules in localized areas of cellular membrane, e.g., in the association of integrins with each other (and other molecules) by lateral interactions. The formation of such clusters may influence various integrin functions in multiple ways, including, for example, by additional or secondary signaling events or interactions, and by altered ligand affinity.
The integrin-mediated function of adhesion is, or various integrin-mediated events associated with adhesion are, important for a variety of physiological and pathological responses. The extent of adhesion is functionally related to integrin signaling. For example, in association with initial integrin-dependent adhesion to a substratum, certain cells change their shape and start spreading on the surface of the stratum, using integrins for establishing new contacts with the underlying proteins (e.g., extracellular matrix (ECM) components). In motile cells, the whole array of integrin-mediated events involving adhesion—initial contact, cell shape change, cell spreading, and cell locomotion—is sometimes termed “the adhesion cascade” (Sharar, S. R., et al., The Adhesion Cascade and Anti-Adhesion Therapy: An Overview, 16 Springer Semin. Immunopathol. 359, 1995). Adhesion cascades are viewed as integral to one or more familiar cell motility patterns, including angiogenesis, lymphocyte homing, tumor cell metastasis, and cell migration processes associated with wound healing, although similar cascade mechanisms are also viewed as operative even in the absence of cell locomotion (e.g., in platelet adhesion and aggregation). Extravasation of neutrophils is described below in greater detail, as a paradigmatic integrin-mediated adhesion cascade (Hub, E., et al., Mechanism of Chemokine-Induced Leukocyte Adhesion and Emigration, Chemoattractant Ligands and Their Receptors (Horuk, R., Ed.), Boca Raton, CRC Press, 1996, 301).
The onset of extravasation is heralded by the appearance in the circulation of chemotactic factors, or chemoattractants (i.e., specific substances that initiate cell migration along their concentration gradients). Chemoattractants (e.g., chemokines, bacterial peptides, and products of complement activation) activate neutrophils to upregulate their integrin receptors (neutrophil integrins include, e.g., LFA-1 [CD11a/CD18], CR3 [also known as Mac-1, CD11b/CD18], and gp150,95 [CD11c/CD18]). Neutrophils thus activated adhere to endotheliocytes, change shape, and spread on the endothelial surface. Thereafter, the stimulated motile apparatus of the neutrophils gives rise to migration, and the neutrophils start moving, first across the endothelial layer and further, through the perivascular ECM, towards the source of the chemotactic stimulus, e.g., pathogenic bacteria invading a certain bodily tissue. During the whole process, from the initial firm contact with the endothelium to the cessation of locomotion at the destination site, various integrins serve to attach the neutrophil to the substrata it encounters, enabling its recruitment to the locus of infection.
Another integrin-mediated function is cell-cell fusion. Under physiological conditions, fusion is a developmentally regulated stage in the differentiation of certain multinucleate cells (e.g., osteoclasts, myocytes, and syncytiotrophoblasts), and fusion is also a prerequisite to fertilization (in the case of sperm-egg fusion). Fusion is effected by specialized cellular systems involving integrins (see, e.g., refs. cited in Huovila, A.-P. J., et al., ADAMs and Cell Fusion, 8 Current Opin. Cell. Biol. 692, 1996 and Ohgimoto, S., et al., Molecular Characterization of Fusion Regulatory Protein-1 [FRP-1] that Induces Multinucleate Giant Cell Formation of Monocytes and HIV gp160-Mediated Cell Fusion: FRP-1 and 4F2/CD98 Are Identical Molecules, 155 J. Immune. 3585, 1995).
The ability to undergo recirculation from intracellular compartments to the cell surface and vice versa is a common property of divers cellular receptors, including integrins (see, e.g., Handagama, P., et al., Kistrin, an Integrin Antagonist, Blocks Endocytosis of Fibrinogen into Guinea-Pig Megakaryocyte and Platelet alpha-Granules, 91 J. Clin. Invest. 193, 1993). This capability of integrins facilitates the mediation of other cellular functions by transporting into the cell extracellular material (e.g., soluble proteins, particulate matter, and other cells). Integrin-mediated internalization is used by certain microorganisms to invade their targets. For example, CR3 mediates entry of iC3b-opsonized HIV-1 and HIV-2 into CD4-negative lymphocytic and monocytic cells (Boyer, V., et al., Complement Mediates Human Immunodeficiency Virus Type 1 Infection of a Human T cell Line in a CD4- and Antibody-Independent Fashion, 173 J. Exp. Med. 1151, 1991).
The above-delineated functions of integrins are illustrative only, as other characterizations of integrin functions can also be made. Moreover, the integrin-mediated functions as delineated herein are overlapping and interrelated. In the case of neutrophil extravasation, for example, the initial chemotactic signal activating the cells is commonly functionally associated with in integrin upregulation (inside-out signaling) and adhesion to the endothelial surface. This adhesion event, in turn, is associated with outside-in signal, enabling the neutrophil to change its shape, which is a prerequisite to the spreading and migration of the cell. Likewise, when the neutrophil that has arrived to the source of chemoattractants establishes an adhesive interaction with the bacteria by means of integrins, an outside-in signal is transduced, which is associated with the initiation of internalization of the integrins involved, together with the bacteria attached thereto (phagocytosis).
Furthermore, regarding outside-in integrin signaling, certain cellular processes are co-mediated by several signaling systems acting in concert. In the case of neutrophils extravasating to the tissues to phagocytose bacteria, the neutrophils receive signals by means of the receptors of the chemoattractant (along the concentration gradient of which the movement occurs) and by means of distinct integrins (those that attach it to the substratum and, subsequently, those recognizing the bacteria). This interplay of signals mediates the antibacterial machinery of the neutrophils with the consequence that only upon contact with the bacteria, which is established by means of a particular type of integrin, are the constituents of the intracellular granules released and reactive oxygen species formed. As a result, the formation and release of microbicidal substances take place preferentially at sites of contact with bacteria, enabling effective killing of the bacteria and preventing the destruction of host tissue (Wright, S. D., Receptors for Complement and the Biology of Phagocytosis [Chapter 25], Inflammation: Basic Principles and Clinical Correlates [Gallin, J. I., et al., Eds.], 2nd Ed., New York, Raven Press, 477, 1992).
Clearly, a broad range of cellular activities can be regulated by modulating certain integrin functions with appropriate agents. One such integrin-modulating agent is ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide):

Ajoene, and a precursor thereof, can be isolated from products derived from extracts of garlic (Allium sativum). As the garlic is crushed, alliin in the garlic comes into contact with alliinase in the cell wall to form allicin. Then, in the presence of a polar solvent, allicin may form ajoene.
Ajoene has been previously shown to inhibit platelet aggregation by inactivating allosterically the platelet integrin, GP IIb/IIIa (Apitz-Castro, R., et al., 141 Biophys. Res. Commun. 145, 1986). It has been demonstrated that stereoisomers of ajoene (ie., E- and Z-4,5,9-trithiadodeca-1,6,1-triene-9-oxides) exhibit no significant differences in their effects on platelets (Block, E., et al., 108 J. Am. Chem. Soc. 7045, 1986). For this reason, most of subsequent studies of the integrin modulation by ajoene were carried out on various mixtures of the E- and Z-isomers. It was shown, for example, that ajoene is a potent inhibitor of a wide variety of adhesion-dependent processes, including neutrophil aggregation, HIV transmission (Tatarintsev, A. V., et al., 6 AIDS 1215, 1992), and tumor metastasis. U.S. Patents issued to Tatarintsev, et al. disclose the use of ajoene for treatment of inflammation (U.S. Pat. No. 5,948,821), arthritis (U.S. Pat. No. 5,856,363), and tumors (U.S. Pat. No. 5,932,621), as well as for contraception (U.S. Pat. No. 5,863,954) and inhibition of immune responses (U.S. Pat. No. 5,863,955). All of these diseases and conditions involve integrin-mediated processes. See also PCT application WO 97/25031, which describes the use of ajoene to treat additional diseases and conditions which involve integrin-mediated processes.
The presence of the sulfoxide group in the molecule of ajoene (a prerequisite to optical isomerism) and stereoisomerism of the compound (around the double bond at carbon 6) create a possibility for four optical isomers (enantiomers): E(R)-, E(S)-, Z(R)-, and Z(S)-4,5,9-trithiadodeca-1,6,11-triene-9-oxides. This possibility has never been suggested in the art. Moreover, in the case of allicin (which also contains a sulfoxide radical), even the existence of optical activity has been questioned, so that the existence of enantiomers, let alone stable enantiomers, would have been considered unlikely (Garlic: The Science and Therapeutic Application of Allium Sativum L. and Related Species, page 56 (Lawson L. D., et al., Eds., 1997)).